Telomerase reverse transcriptase-based therapies

ABSTRACT

The invention provides compositions and methods useful for the treatment and prevention of conditions associated with short telomere length.

FIELD OF INVENTION

This invention falls within the field of molecular biology,biotechnology and medicine. More particularly, it relates tocompositions and methods useful for the treatment of conditionsassociated with short telomere length. More particularly, it relates tocompositions and methods useful for the treatment of conditionsassociated with aplastic anemia.

BACKGROUND OF THE INVENTION

Telomeres are specialized structures at the ends of chromosomes, whichhave a role in protecting the chromosome ends from DNA repair anddegrading activities (Blackburn, 2001. Cell 106, 661-673; de Lange,2005. Genes Dev. 19, 2100-2110) Mammalian telomeres consist of TTAGGGrepeats bound by a multi-protein complex known as shelterin (de Lange,2005. Genes Dev. 19, 2100-2110). A minimum length of TTAGGG repeats andthe integrity of the shelterin complex are necessary for telomereprotection (Blackburn, 2001. Cell 106, 661-673; de Lange, 2005. GenesDev. 19, 2100-2110). Telomerase is a cellular reverse transcriptase(TERT, telomerase reverse transcriptase; also known as TP2; TRT; EST2;TCSl; hEST2) capable of compensating telomere attrition through de novoaddition of TTAGGG repeats onto the chromosome ends by using anassociated R A component as template (Terc, telomerase RNA component)(Greider and Blackburn, 1985. Cell 43, 405-413). Telomerase is expressedin most adult stem cell compai tments, however, this is not sufficientto maintain telomere length as evidenced by the fact that telomereshortening occurs with age in most human and mouse tissues (Harley etal., 1990. Nature 345, 458-460; Blasco, 2007. Nat Chem Biol. 3, 640-649;Flores et al, 2008. Genes and Dev 22, 654-667).

Mice carrying homozygous deletion for the TERC gene (the telomerase RNAcomponent) lack any detectable telomerase activity and showedprogressive telomere shortening from one generation to the other at arate comparable to the rate reported in human cells (Blasco et al.,1997). Severe phenotypes typical of late generation TERC−/− mice (e.g.bone marrow aplasia and signs of premature aging) could be rescued byre-introducing a copy of the TERC gene (Samper et al., 2001). Multipletissue degeneration arising in later generations in a conditional mousemodel defective for TERT (the catalytic telomerase subunit) could bereversed upon telomerase reactivation even in aged mice (Jaskelioff etal., 2011).

In the context of wild-type mice, introducing an additional copy of thetelomerase gene, which is expressed in a wide range of epithelialtissues, led to an increased wound healing capacity of the skin(Gonzalez-Suarez et al., 2001). When this allele was introduced in atumour-resistant genetic background (Sp53/Sp16/SArf) remarkable delay ofaging in concert with an increased median lifespan of 40% compared tomice not expressing the telomerase transgene was observed (Tomas-Loba etal., 2008).

A virus (AAV) based telomerase gene therapy was found to be beneficialto extend health span, in the context of normal physiological aging inwild-type mice. In the study examining this benefit, adult and aged micewere subjected to AAV9-mTERT gene therapy to broadly express thecatalytic subunit of mouse telomerase (mTERT). The health span of theTERT treated mice was significantly increased, and aging wasdecelerated, as indicated by a number of physiological parameters(glucose and insulin tolerance, osteoporosis, neuromuscularcoordination, rota-rod, etc). In addition, their mean lifespan, comparedto control groups, was increased by 24% and 13% in adult an old mice,respectively. A single intravenous administration of AAV9-TERT in adultmice resulted in an increase in telomere length in peripheral bloodcells (Bernardes de Jesus et al., 2012).

Shortened telomeres have been associated with numerous diseases, such asDyskeratosis congenita, Aplastic anaemia, Myelodysplastic Syndrome, andFanconi anaemia. Given the severity of these diseases and the poorprognosis of the patients suffering from them, there is a need for noveltherapies to treat diseases associated with short telomere length.

Aplastic anemia is a potentially life-threatening, rare andheterogeneous disorder of the blood in which the bone marrow cannotproduce sufficiently enough new blood cells due to a marked reduction ofimmature hematopoietic stem (HSC) and progenitor cells (Scopes et al.,1994, Maciejewski et al., 1994). Accordingly, the main diseasemanifestations are pancytopenia and marrow hypoplasia which can emergeat any stage of life but are more frequent in young people (age 10-25years) and the elderly (>60 years) (Marsh et al., 2009). Aplastic anemiacan be acquired or inherited. The acquired type is mainlyautoimmune-mediated but can also be triggered by environmental factorssuch as radiation, toxin and virus exposure (Nakao, 1997). Thecongenital form is rarer, however, mutations in more than 30 genes withfunctions in DNA repair, ribosome biogenesis and telomere maintenancepathways have been identified to date (Dokal & Vulliamy, 2010). Afrequently observed clinical feature of aplastic anemia is shorttelomere length in peripheral blood leukocytes even in the absence ofmutations in the telomere maintenance machinery.

Telomeres, the termini of vertebrate chromosomes are highly specialisednucleoprotein structures composed of hexanucleotide (TTAGGG) tandemrepeat sequences which are bound by a six protein complex (TRF1, TRF2,TIN2, RAP1, TPP1 and POT1) termed shelterin (Blackburn, 2001, de Lange,2005). These structures are essential for chromosome integrity bypreventing telomere fusions and telomere fragility. Telomere length iscontrolled by the ribonucleoprotein enzyme telomerase which can de novoadd telomeric sequences onto telomeres. Because telomeric sequence isnaturally lost upon every cell division (known as the end replicationproblem) and somatic cells express telomerase at very low levels or notat all telomeres shorten throughout life. When telomeres becomecritically short they lose their protective function and a persistentDNA damage response at the telomeres is triggered which subsequentlyleads to a cellular senescence response (Harley et al., 1990, Flores etal., 2008). HSCs, in contrast to most somatic cells, show low level oftelomerase activity. However, this activity is insufficient to stoptelomere attrition and consequently the regeneration potential of HSCscells may become limited during the aging process (Hiyama & Hiyama,2007). In line with this, recipients of bone marrow transplants haveshorter telomere lengths than their donors suggesting that telomerasecannot cope with increased replicative proliferation demand during theengraftment phase (Wynn et al., 1998). Moreover, telomeres have beenshown to shorten much faster in patients with aplastic anemia comparedto the normal aging-related attrition found in healthy individualspotentially owed to a higher than normal number of cell divisions (Ballet al., 1998).

Accelerated telomere shortening due to defects in telomere components ortelomerase itself prematurely limits the proliferation potential ofcells which particularly affects the tissue renewal capacity in stemcell compartments (Harley et al., 1990, Flores et al., 2005). Thus,tissues with a high proliferative index such as the hematopoietic systemare particularly affected by lower than normal telomerase levels whichcan ultimately lead to severe disorders such as aplastic anemia(Vulliamy et al., 2002). For instance, the telomeropathy dyskeratosiscongenita has been linked to mutations in 7 genes with importantfunctions in telomere maintenance (TERT, TERC, DKC1, TIN2, NOP10, NHP2and TCAB1) and is characterized by very short telomeres. Dyskeratosiscongenita is a multisystem syndrome comprising diverse clinical featuressuch as nail dystrophy, oral leucoplakia, abnormal skin pigmentation andcerebellar hypoplasia (Dokal, 2011). The most severe complication,however, is the development of aplastic anemia in 80% of the casesunderlining that the clinical features are caused by excessive telomereshortening which eventually leads to the exhaustion of the stem cellreserve (Dokal & Vulliamy, 2010).

The causality between proliferation potential and telomere lengthsuggests that a therapeutic intervention with telomerase, aimed atpreventing telomere loss beyond a critically short length, may be afeasible strategy to treat those forms of aplastic anemia associatedwith limited blood forming capacity due to the presence of shorttelomeres. In this regard, we previously developed a telomerase (Tert)gene therapy using adeno-associated virus (AAV9) vectors. Interestingly,telomerase gene therapy using AAV9 Tert in adult wilt-type miceattenuated or reverted the aging-associated telomere erosion inperipheral blood monocytes (Bernardes de Jesus et al., 2012), suggestingthat this gene therapy may be effective in the treatment ofhematological disorders related to short telomeres.

To test this hypothesis we used our recently generated mouse model ofaplastic anemia which recapitulates the bone marrow phenotype observedin patients (Beier et al., 2012). In this mouse model bone marrowspecific depletion of the shelterin gene Trfl cause severe telomereuncapping and provokes a DNA damage response which in turn leads to afast clearance of those HSCs and progenitor cells deficient for Trfl.However, in this model we induce Trfl deletion at a frequency that doesnot target 100% of the HSCs and progenitor cells. Therefore, cells thatretain intact Trfl undergo additional rounds of compensatoryproliferation leading to fast telomere attrition. Thus, partialdepletion of the stem and progenitor cell compartment by Trfl deletionrecapitulates the compensatory hyperproliferation observed after bonemarrow transplantation or in autoimmune-mediated aplastic anemia, aswell as presence of very short telomeres in patients owing to mutationsin telomere maintenance genes. Interestingly, in our mouse model we canadjust the rate of telomere shortening through the frequency of Trfldeletion-mediated HSC depletion which allows to control the onset ofbone marrow aplasia and pancytopenia (Beier et al., 2012).

In this study we employ this mouse model of aplastic anemia toinvestigate whether telomerase activation using state of the art genetherapy vectors can be an effective treatment to attenuate telomereattrition and HSC depletion, and thus prevent bone marrow failure.

SUMMARY

The invention provides compositions and methods useful for the treatmentand prevention of conditions associated with short telomere length.

One aspect of the invention provides a method of treating a patient witha condition associated with short telomere length comprisingadministering to the patient a nucleic acid vector comprising a codingsequence for telomerase reverse transcriptase (TERT). In one embodiment,the TERT is encoded by a nucleic acid sequence comprising a sequencethat is at least 90% identical to the sequence of SEQ ID NO: 1 or SEQ IDNO: 3. In one embodiment, the TERT is encoded by a nucleic acid sequencecomprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In oneembodiment, the TERT is encoded by a nucleic acid sequence consisting ofthe sequence of SEQ ID NO: 1 or SEQ ID NO: 3 In one embodiment, the TERTcomprises an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO:2 or SEQ ID NO: 4. In one embodiment,the TERT comprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In one embodiment, the TERT consists of the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO: 4. In one embodiment, the nucleic acidsequence encoding TERT is operably linked to a regulatory sequence thatdrives the expression of the coding sequence. In one embodiment, thevector is a non-integrative vector, such as an adeno-associatedvirus-based non-integrative vector. In one embodiment, the vector is anadeno-associated virus-based vector derived from a serotype 9adeno-associated virus (AAV9). In one embodiment, the capsid of theadeno-associated virus-based vector is made of capsid proteins of theserotype 9 adeno-associated virus (AAV9), and the nucleic acid sequencecontained in the capsid is flanked at both ends by internal terminalrepeats corresponding to serotype 2 adenoassociated viruses. In oneembodiment, the nucleic acid contained in the capsid comprises afragment which encodes the amino acid sequence coding for TERT. In oneembodiment, the vector comprises a regulatory sequence which is aconstitutive promoter. In one embodiment, the regulatory sequence is thecytomegalovirus (CMV) promoter. In one embodiment, the conditionassociated with short telomere length is characterized by mutations in agene or genes involved in telomere maintenance. In one embodiment, thecondition associated with short telomere length is selected from thegroup consisting of Dyskeratosis congenita, Aplastic anaemia,Myelodysplastic Syndrome, Fanconi anaemia.

In yet further embodiments, the invention is directed to the followingset of subject matters:

1. A method of treating a patient with a condition associated with shorttelomere length comprising administering to the patient a nucleic acidvector comprising a coding sequence for telomerase reverse transcriptase(TERT).

2. The method of 1, wherein TERT is encoded by a nucleic acid sequencecomprising a sequence that is at least 90% identical to the sequence ofSEQ ID NO: 1 or SEQ ID NO: 3.

3. The method of 1 or 2, wherein TERT is encoded by a nucleic acidsequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

4. The method of any of 1-3, wherein TERT is encoded by a nucleic acidsequence consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3

5. The method of any of 1-4, wherein TERT comprises an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO: 4.

6. The method of any of 1-5, wherein TERT comprises an amino acidsequence of SEQ ID NO:2 or SEQ ID NO: 4.

7. The method of any of 1-6, wherein TERT consists of the amino acidsequence of SEQ ID NO:2 or SEQ ID NO: 4.

8. The method of any of 1-7, wherein the nucleic acid sequence encodingTERT is operably linked to a regulatory sequence that drives theexpression of the coding sequence.

9. The method of any of 1-8, wherein the vector is a non-integrativevector.

10. The method of any of 1-9, wherein the vector is an adeno-associatedvirus-based non-integrative vector.

11. The method of any of 1-10, wherein the vector is an adeno-associatedvirus-based vector derived from a serotype 9 adeno-associated virus(AAV9).

12. The method of 11, wherein the capsid of the adeno-associatedvirus-based vector is made of capsid proteins of the serotype 9adeno-associated virus (AAV9), and the nucleic acid sequence containedin the capsid is flanked at both ends by internal terminal repeatscorresponding to serotype 2 adenoassociated viruses.

13. The method of 12, wherein the nucleic acid contained in the capsidcomprises a fragment which encodes the amino acid sequence coding forTERT.

14. The method of any of 1-13, wherein the vector comprises a regulatorysequence which is a constitutive promoter.

15. The method of 14, wherein the regulatory sequence is thecytomegalovirus (CMV) promoter.

16. The method of any of 1-15, wherein the condition associated withshort telomere length is characterized by mutations in a gene or genesinvolved in telomere maintenance.

17. The method of any of 1-16, wherein the condition associated withshort telomere length is selected from the group consisting ofDyskeratosis congenita, Aplastic anaemia, Myelodysplastic Syndrome,Fanconi anaemia, and pulmonary fibrosis.

18. A nucleic acid vector comprising a coding sequence for telomerasereverse transcriptase (TERT) for use in treating a condition associatedwith with short telomere length.

19. The nucleic acid vector of 18, wherein TERT is encoded by a nucleicacid sequence comprising a sequence that is at least 90% identical tothe sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

20. The nucleic acid vector of 18 or 19, wherein TERT is encoded by anucleic acid sequence comprising the sequence of SEQ ID NO: 1 or SEQ IDNO: 3.

21. The nucleic acid vector of any of 18-20, wherein TERT is encoded bya nucleic acid sequence consisting of the sequence of SEQ ID NO: 1 orSEQ ID NO: 3

22. The nucleic acid vector of any of 18-21, wherein TERT comprises anamino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO:2 or SEQ ID NO: 4.

23. The nucleic acid vector of any of 18-22, wherein TERT comprises anamino acid sequence of SEQ ID NO:2 or SEQ ID NO: 4.

24. The nucleic acid vector of any of 18-23, wherein TERT consists ofthe amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 4.

25. The nucleic acid vector of any of 18-24, wherein the nucleic acidsequence encoding TERT is operably linked to a regulatory sequence thatdrives the expression of the coding sequence.

26. The nucleic acid vector of any of 18-25, wherein the vector is anon-integrative vector.

27. The nucleic acid vector of any of 18-26, wherein the vector is anadeno-associated virus-based non-integrative vector.

28. The nucleic acid vector of any of 18-27, wherein the vector is anadeno-associated virus-based vector derived from a serotype 9adeno-associated virus (AAV9).

29. The nucleic acid vector of 28, wherein the capsid of theadeno-associated virus-based vector is made of capsid proteins of theserotype 9 adeno-associated virus (AAV9), and the nucleic acid sequencecontained in the capsid is flanked at both ends by internal terminalrepeats corresponding to serotype 2 adenoassociated viruses.

30. The nucleic acid vector of 29, wherein the nucleic acid contained inthe capsid comprises a fragment which encodes the amino acid sequencecoding for TERT.

31. The nucleic acid vector of any of 18-30, wherein the vectorcomprises a regulatory sequence which is a constitutive promoter.

32. The nucleic acid vector of 31, wherein the regulatory sequence isthe cytomegalovirus (CMV) promoter.

33. The nucleic acid vector of any of 18-32, wherein the conditionassociated with short telomere length is characterized by mutations in agene or genes involved in telomere maintenance.

34. The nucleic acid vector of any of 18-33, wherein the conditionassociated with short telomere length is selected from the groupconsisting of Dyskeratosis congenita, Aplastic anaemia, MyelodysplasticSyndrome, Fanconi anaemia, and pulmonary fibrosis.

35. The nucleic acid vector of any of 18-34, wherein the conditionassociated with short telomere length is dyskeratosis congenita.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: AAV9-Tert effects on survival (A-C) and blood counts (D-E)

FIG. 2: AAV9-Tert effects on telomere length in peripheral blood (A-C)and bone marrow (D-E)

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The invention provides compositions and methods useful for the treatmentand prevention of conditions associated with short telomere length.

A “condition associated with short telomere length” is one which ischaracterized by an accumulation of critically short telomeres. Incertain embodiments, subjects suffering from such a condition exhibitpremature onset of pathologies resulting from a defective regenerativecapacity of tissues.

In certain embodiments, the condition associated with short telomerelength is characterized by mutations in a gene or genes involved intelomere maintenance. Specific examples of such genetically basedconditions include, but are not limited to Dyskeratosis congenita,Aplastic anaemia, Myelodysplastic Syndrome, Fanconi anaemia, andpulmonary fibrosis.

Dyskeratosis congenita (DKC) is a genetically heterogeneous humandisease, which is paradigmatic of premature ageing syndromes (Dokal,2011). DKC is characterised by the presence of short/dysfunctionaltelomeres owing to mutations in genes related to telomere maintenance,being the most frequently mutated those encoding proteins of thetelomerase complex (i.e. TERT, TERC, NOP10, DKC1, NHP2) (Dokal, 2011;Dokal and Vulliamy, 2010; Mason and Bessler, 2011; Savage and Alter,2008). In addition, a subset of patients carry mutations in the geneencoding TIN2 (TRF1-interacting protein) a component of the shelterincomplex, which binds and protects mammalian telomeres (Dokal, 2011;Martinez and Blasco, 2011; Walne et al., 2008). Both a functionaltelomerase complex and a proper telomere capping structure by theshelterin proteins are required for maintenance and capping ofchromosome ends, respectively.

Clinical features of patients suffering from DKC include skinabnormalities (i.e. skin hyperpigmentation), signs of premature aging(i.e. hair greying, nail dystrophy, oral leucoplakia, etc),predisposition to cancer, and several other life-threatening conditions,including aplastic anemia and pulmonary fibrosis (Armanios andBlackburn, 2012). In particular, tissues with a high proliferative indexare most affected due to the loss of telomeric DNA that occurs upon eachcell division. This explains why DKC patients are particularlyvulnerable to impaired bone marrow function leading to pancytopenia andeventually bone marrow failure (BMF) (Armanios and Blackburn, 2012;Blasco, 2007)

Aplastic anaemia, is a life threatening bone marrow disordercharacterised by hypocellular bone marrow and low blood cell counts.Patients with acquired aplastic anaemia present with leukocytes whichhave considerably shorter telomeres than age-matched healthy individuals(Carroll and Ly, 2009). Aplastic anaemia is frequently caused by anautoimmune mediated attack against hematopoietic stem cells. However,recent studies demonstrated that mutations in the core telomerasecomponents TERT and TERC are the underlying cause in a clinicallyrelevant subpopulation (Yamaguchi et al., 2003; Yamaguchi et al., 2005).Mutations in the core telomerase components TERT and TERC, as well as inthe shelterin component TIN2 have been linked to this disease (Savage etal., 2006).

Myelodysplastic Syndrome (MDS) encompasses several bone marrow diseasescharacterised by ineffective production of the myeloid class of bloodcells. Caused by progressive bone marrow failure, similar to DKC, MDSpatients often report with severe anaemia and cytopenias. Inapproximately one third of the cases the disease progresses quickly andtransforms into acute myelogenous leukemia (AML) which is particularlyresistant to treatment. Even though shortened telomeres in patients withMDS suggest that insufficient or hampered telomeric maintenance iscausative for the syndrome, only 3 out of 210 cases showed heterozygousTERC mutations in a previous study (Yamaguchi et al., 2003). However, arecently published study clearly circumstantiated the connection betweenhuman telomerase mutations and MDS, aplastic anaemia and AML. (Holme etal., 2012) reported various families with mutations of the telomerasecomponents TERC and TERT which e.g. the grandfather suffered from AML,the daughter from MDS and the grandson from aplastic anaemia (Holme etal., 2012) emphasising the close relation of different clinicalmanifestations with impaired telomere maintenance.

Fanconi anaemia (FA) is a heterogeneous genetic disease caused bymutations in genes involved in DNA repair. Affected individuals displaymultiple congenital defects and haematological deficiencies at a youngage of onset (Kee and D'Andrea, 2012). Manifestations related to thelatter however are the predominant symptoms of this syndrome and as thedisease progresses can develop into the aforementioned syndromesincluding aplastic anaemia, MDS and AML. Importantly, patients sufferingfrom FA have been also shown to present shorter telomeres than normal(Gadalla et al., 2010). The facts that mutations causing FA showimpaired DNA damage response (DDR) and telomeres are particularlyvulnerable to replicative stress may provide an explanation for theobserved telomere erosion. In support of this Callen et al. (2002)suggested that in FA patients increased telomere breakage in concertwith replicative shortening account for the observed telomereshortening.

Pulmonary fibrosis refers to a condition characterised by scarring ofthe lung tissue. Pulmonary fibrosis can be caused by many factors,including chronic inflammatory processes, infections, environmentalcompounds, ionizing radiation (for example radiation therapy to treattumors of the chest), chronic medical conditions (lupus, rheumatoidarthritis). Idiopathic pulmonary fibrosis (IPF) refers to pulmonaryfibrosis without an identifiable cause.

Accordingly, the invention provides methods of treating a patientsuffering from a condition associated with short telomere lengthcomprising administering to the patient an agent which increases thetelomere length of the patient. In one embodiment, the agent preventsdegradation of the chromosomal ends. In one embodiment, the agentincreases the activity of telomerase reverse transcriptase (TERT). Inone embodiment, the method of treatment is a gene therapy methodcomprises administering to the patient a nucleic acid vector comprisinga coding sequence for telomerase reverse transcriptase (TERT).

In certain embodiments, the TERT sequence used in the gene therapyvector is derived from the same species as the subject. For example,gene therapy in humans would be carried out using the human TERTsequence. Gene therapy in mice would be carried out using the mouse TERTsequence, as described in the examples. In one embodiment, the TERT isencoded by the nucleic acid sequence as set forth in SEQ ID NO: 1 or SEQID NO: 3 (human TERT variants 1 and 2), or is an active fragment orfunctional equivalent of SEQ ID NO: 1 or SEQ ID NO: 3. The polypeptidesequence encoded by SEQ ID NO: 1 is set forth in SEQ ID NO: 2. Thepolypeptide encoded by SEQ ID NO: 3 is set forth in SEQ ID NO: 4. Asused herein, “functional equivalent” refers to a nucleic acid moleculethat encodes a polypeptide that has TERT activity or a polypeptide thathas TERT activity. The functional equivalent may displays 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, 100% or more activity compared to TERT encodedby SEQ ID NO: 1 or SEQ ID NO: 3. Functional equivalents may beartificial or naturally-occurring. For example, naturally-occurringvariants of the TERT sequence in a population fall within the scope offunctional equivalent. TERT sequences derived from other species alsofall within the scope of the term “functional equivalent”, in particularthe murine TERT sequence given in SEQ ID NO: 5. In a particularembodiment, the functional equivalent is a nucleic acid with anucleotide sequence having at least 75%, 80%>, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.9% identity to SEQ ID NO: 1 or SEQ ID NO: 3. In afurther embodiment, the functional equivalent is a polypeptide with anamino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.9% identity to SEQ ID NO: 2 or SEQ ID NO: 4. In thecase of functional equivalents, sequence identity should be calculatedalong the entire length of the nucleic acid. Functional equivalents maycontain one or more, e.g. 2, 3, 4, 5, 10, 15, 20, 30 or more, nucleotideinsertions, deletions and/or substitutions when compared to SEQ ID NO: 1or SEQ ID NO: 3. The term “functional equivalent” also encompassesnucleic acid sequences that encode a TERT polypeptide with at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identityto the sequence as set forth in SEQ ID NO:2 or SEQ ID NO: 4, but thatshow little homology to the nucleic acid sequence given in SEQ ID NO: 1or SEQ ID NO: 3 because of the degeneracy of the genetic code.

As used herein, the term “active fragment” refers to a nucleic acidmolecule that encodes a polypeptide that has TERT activity orpolypeptide that has TERT activity, but which is a fragment of thenucleic acid as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 or the aminoacid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4. An activefragment may be of any size provided that TERT activity is retained. Afragment will have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,99.5%, 100% identity to SEQ ID NO: 1-4 along the length of the alignmentbetween the shorter fragment and SEQ ID NO: 1-4.

Fusion proteins including these fragments can be comprised in thenucleic acid vectors needed to carry out the invention. For example, anadditional 5, 10, 20, 30, 40, 50 or even 100 amino acid residues fromthe polypeptide sequence, or from a homologous sequence, may be includedat either or both the C terminal and/or N terminus without prejudicingthe ability of the polypeptide fragment to fold correctly and exhibitbiological activity.

Sequence identity may be calculated by any one of the various methods inthe art, including for example BLAST (Altschul S F, Gish W, Miller W,Myers E W, Lipman D J (1990). “Basic local alignment search tool”. J MolBiol 215 (3): 403-410) and FASTA (Lipman, D J; Pearson, W R (1985).“Rapid and sensitive protein similarity searches”. Science 227 (4693):1435-41; http://fasta.bioch.Virginia,edu/fasta_www2/fasta_list2.shtml)and variations on these alignment programs.

In one embodiment, the method of treatment is a gene therapy methodand/or the nucleic acid vector used is a gene therapy vector. Genetherapy methods and vectors are well known in the art and generallycomprise delivering a nucleic acid encoding a therapeutically activeprotein to a subject. The nucleic acid may be delivered in a number ofways including delivering naked DNA such as plasmid or mini-circles, theuse of liposomes or cationic polymers or other engineered nano-particlescontaining the nucleic acid, or viral vectors that encapsidate thenucleic acid.

In a further embodiment, the gene therapy is achieved using stabletransformation of organisms with an inducible expression system.Suitable inducible expression systems are known in the art and includethe CRE-LOX recombinase based system which is suitable for use in miceand tetracycline-regulated which can be used in the treatment of humansubjects.

In one embodiment the gene therapy vector is a viral vector. Viral genetherapy vectors are well known in the art. Vectors include integrativeand non-integrative vectors such as those based on retroviruses,adenoviruses (AdV), adeno-associated viruses (AAV), lentiviruses, poxviruses, alphaviruses, and herpes viruses.

Using non-integrative viral vectors, such as AAV, seems to beparticularly advantageous. In one aspect, this is becausenon-integrative vectors do not cause any permanent genetic modification.Second, the vectors target to adult tissues, avoiding having thesubjects under the effect of constitutive telomerase expression fromearly stages of development. Additionally, non-integrative vectorseffectively incorporate a safety mechanism to avoid over-proliferationof TERT expressing cells. Cells will lose the vector (and, as aconsequence, the telomerase expression) if they start proliferatingquickly.

Particular examples of suitable non-integrative vectors include thosebased on adenoviruses (AdV) in particular gutless adenoviruses,adeno-associated viruses (AAV), integrase deficient lentiviruses, poxviruses, alphaviruses, and herpes viruses. Preferably, thenon-integrative vector used in the invention is an adeno-associatedvirus-based non-integrative vector, similar to natural adeno-associatedvirus particles. AAV preferentially targets post-mitotic tissues, whichare considered more resistant to cancer than the highly proliferativeones. Examples of adeno-associated virus-based non integrative vectorsinclude vectors based on any AAV serotype, i.e. AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and pseudotyped AAV. Tissuespecificity is determined by the capsid serotype. Pseudotyping of AAVvectors and capsid engineering to alter their tropism range will likelybe important to their use in therapy.

Vectors derived from adeno-associated viruses (AAVs) have emerged as oneof the vectors of choice for many gene transfer applications because oftheir many desirable properties, including capability to transduce abroad range of tissues at high efficiency, poor immunogenicity and anexcellent safety profile (Merten, Geny-Fiamma et al. 2005; Buning,Perabo et al. 2008), toxicity being absent in many preclinical models(Niemeyer, Herzog et al Blood 2009; Mas, Montane et al Diabetes 2006;Jiang, Lillicrap et al blood 2006; Ghosh, Yue et al Molecular therapy2007; Tafuro, Ayuso et al cardiovascular research 2009). AAV vectorstransduce post-mitotic cells and can sustain long-term gene expression(up to several years) both in small and large animal models of disease(Niemeyer, Herzog et al Blood 2009; Mas, Montane et al Diabetes 2006;Jiang, Lillicrap et al blood 2006; Ghosh, Yue et al Molecular therapy2007; Tafuro, Ayuso et al cardiovascular research 2009). Safety andefficacy of AAV gene transfer has been extensively studied in humanswith encouraging results in the liver, muscle, CNS, and retina (Manno etal Nat medicine 2006, Stroes et al ATVB 2008, Kaplitt, Feigin, Lancet2009; Maguire, Simonelli et al NEJM 2008; Bainbridge et al NEJM 2008).

AAV2 is the best characterized serotype for gene transfer studies bothin humans and experimental models. AAV2 presents natural tropism towardsskeletal muscles, neurons, vascular smooth muscle cells and hepatocytes.AAV2 is therefore a good choice of vector to target these tissues, inparticular when using the methods or vectors of the invention to treat acondition associated with one of these tissues. For example, treatmentof neuromuscular degeneration may be targeted to skeletal muscle and/orneurons in this way.

Newly isolated serotypes, such as AAV7, AAV8, and AAV9 have beensuccessfully adopted in preclinical studies (Gao, Alvira et al PNAS2002). Although limited immunologic responses have been detected inhuman subjects treated with AAV2 or AAV1 against the AAV capsid (Mannoet al Nat Med 2006; Mingozzi et al Nat Med 2007; Brantly et al PNAS2009; Mingozzi et al blood 2009), long term expression of thetherapeutic gene is possible depending on the target tissue and theroute of administration (Brantly et al PNAS 2009; Simonelli et al moltherapy 2010). In addition, the use of non-human serotypes, like AAV8and AAV9, might be useful to overcome these immunological responses insubjects, and clinical trials have just commenced (ClinicalTrials.govIdentifier: NCT00979238). Altogether, these encouraging data suggestthat AAV vectors are useful tools to treat human diseases with a highsafety and efficient profile.

The choice of adeno-associated viruses of wide tropism, such as thosederived from serotype 9 adeno-associated virus (AAV9) is particularlyadvantageous when treating conditions associated with short telomerelength. AAV9 viruses have shown efficient transduction in a broad rangeof tissues, with high tropism for liver, heart and skeletal muscle(Inagaki et al Molecular Therapy 2006) and thus the beneficial effectsof gene therapy can be achieved in more tissues. In addition, AAV9vectors have the unique ability to cross the blood-brain-barrier andtarget the brain upon intravenous injection in adult mice and cats(Foust et al Nature biotechnology 2009; Duque et al Molecular therapy etal 2009).

One aspect of the invention provides a system in which the capsid (whichis the part of the virus which determines the virus tropism) of theadeno-associated virus-based vector is made of capsid proteins of theserotype 9 adeno-associated virus (AAV9). In one embodiment of the viralvectors for use in the invention, the polynucleotide sequence packed inthe capsid is flanked by internal terminal repeats (ITRs) of anadeno-associated virus, preferably of serotype 2 which has beenextensively characterised in the art, and presents a coding sequencelocated between the ITRs. As set out above, the nucleic acid preferablycodes for a functional TERT polypeptide. In one embodiment, theregulatory sequence operatively linked to the TERT coding sequence isthe cytomegalovirus promoter (CMV), although other suitable regulatorysequences will be known to those of skill in the art.

When treating conditions associated with short telomere length, it isadvantageous to target the treatment to the effected tissues. The choiceof AAV serotype for the capsid protein of the gene therapy vector may bethus based on the desired site of gene therapy. If the target tissue isskeletal muscle, for example, in treating loss of neuromuscularcoordination, AAV1- and AAV6-based viral vectors can be used. Both ofthese serotypes are more efficient at trans fecting muscle than otherAAV serotypes. AAV3 is useful for trans fecting haematopoietic cells. Athorough review of AAV-based vectors for gene therapy can be found inShi et al, (2008) “AAV-based targeting gene therapy” Am. J. Immunol.4:51-65.

Alternatively, other viral vectors can be used in the present invention.Any vector compatible with use in gene therapy can be used in thepresent invention. Heilbronn & Weger (2010) Handb Exp Pharmacol. 197:143-70 provides a review of viral vectors that are useful in genetherapy. In accordance with all the previous discussion, vectorscomprising a coding sequence for telomerase reverse transcriptase (TERT)suitable for use in gene therapy are an important point for putting theinvention into practice. Suitable gene therapy vectors include any kindof particle that comprises a polynucleotide fragment encoding thetelomerase reverse transcriptase (TERT) protein, operably linked to aregulatory element such as a promoter, which allows the expression of afunctional TERT protein demonstrating telomerase reverse transcriptaseactivity in the targeted cells. Preferably, TERT is encoded by thenucleic acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, oris an active fragment or functional equivalent of TERT.

The term gene therapy vector includes within its scope naked DNAmolecules such as plasmids or mini-circles, i.e. circular DNA moleculeswhich do not contain bacterial DNA sequences, provided that the TERTcoding sequence and its linked regulatory element are inserted in theplasmid, as well as to more complicated systems, such as particles withthe structure of virions (viral particles), comprising at least a capsidand at least a polynucleotide sequence, with a size that allows thepolynucleotide sequence to be packed within the capsid in a mannersimilar to that of the native genome of the virus of origin of thecapsid. The polynucleotide sequence must include a region where the TERTcoding sequence and its linked regulatory element are inserted such thatthe telomerase reverse transcriptase protein can be expressed from thatpolynucleotide sequence once the viral particle has infected a cell.

In one embodiment, the gene therapy vector suitable for being used inthe invention is a non-integrative vector, such as an adeno-associatedvirus-based non-integrative vector. For the purposes of the invention,the choice of non-integrative vectors seems to be particularlyadvantageous, because they do not cause any permanent geneticmodification. Also, as stated before, such vectors incorporate a safetymechanism to avoid over-proliferation of TERT expressing cells that willlose the vector if the cells start proliferating quickly.

Adeno-associated virus-based vectors derived from a serotype 9adeno-associated virus (AAV9) are preferred because the beneficialeffects can be achieved in more tissues (see above). In one particularlypreferred embodiment, the regulatory sequence operatively linked to theTERT coding sequence is the cytomegalovirus promoter (CMV). The nucleicacid sequence encoding TERT is operably linked to a regulatory sequencethat drives the expression of the coding sequence. As used herein, theterm “regulatory element” means a nucleic acid sequence that serves as apromoter, i.e., regulates expression of a nucleic acid sequence operablylinked to the promoter. Such “regulatory elements” or “promoters” cancontrol the expression of linked nucleic acid sequences eitherconstitutively or inducible.

The regulatory sequence may be a constitutive promoter. An example of aregulatory sequence which is a constitutive promoter is thecytomegalovirus (CMV) promoter.

The expression of TERT following gene therapy according to the inventionpersists for a time of several months to several years. In mice, TERTexpression was detectable after 5 months. In monkey, gene expressionfollowing gene therapy with an AAV-based vector has been detected up to6 years after treatment and up to 8 years in dogs (Rivera et al Blood2005, and Niemeyer et al blood 2009). Frequent repetition of treatmentusing the methods and vectors of the invention is therefore notnecessary. In one embodiment of the invention, the subject is treatedonce. In an alternative embodiment, the subject is treated initially,and is then treated again once TERT expression levels decrease by about50% of those attained immediately following treatment. Treatment may berepeated with the same or alternative vector to maintain the reductionin age-related disorders if necessary, for example annually, or onceevery 5 years or once a decade. When administering a second orsubsequent dose, it may be necessary to use a different gene therapyvector, for example when using an AAV-based vector the second andsubsequent administrations may be a vector with a capsid derived from adifferent serotype than that used for the first administration. It ispossible that a subject may develop neutralising antibodies to the firstgene therapy vector, making it ineffective if administered a second orsubsequent time (Amado et al (2010) Science Translational Medicine2(21):21ra16).

The methods of treatment of the invention have the effect of treatingand/or preventing conditions associated with short telomere length. In afurther aspect, therefore, the invention refers to a gene therapy methodor the use of a nucleic acid vector as described above, for use in thetreatment or prevention in a subject of condition associated with shorttelomere length, including but not limited to genetically basedconditions such as Dyskeratosis congenita, Aplastic anaemia,Myelodysplastic Syndrome, Fanconi anaemia, and pulmonary fibrosis.

The effectiveness of treatment of the conditions associated with shorttelomere length can be measured by various methods known in the art. Inone embodiment, the effectiveness of the treatment is measured by anincrease in lifespan of a treated patient suffering from a conditionassociated with short telomere length as compared to the expectedlifespan of an untreated patient suffering from the same condition. Incertain embodiments, the lifespan is extended by 5%, 10%, 15%, 20% ormore, with reference to the expected lifespan for a patient sufferingfrom the same condition.

In one embodiment, the effectiveness of the treatment is measured by adelayed or prevented bone marrow failure in a treated patient sufferingfrom a condition associated with short telomere as compared to theexpected onset of bone marrow failure in an untreated patient sufferingfrom the same condition. In certain embodiments, the delay in the onsetof bone marrow failure of a treated patient suffering from a conditionassociated with short telomere length is extended by 5%, 10%, 15%, 20%or more, with reference to the expected onset of bone marrow failure foran untreated patient suffering from the same condition.

In one embodiment, the effectiveness of the treatment is measured by anincrease in overall fitness of a treated patient suffering from acondition associated with short telomere length treated as compared tothe overall fitness of an untreated patient suffering from the samecondition. Overall fitness can be determined by measuring physicalattributes associated with the particular condition. Examples of suchphysical attributes include skin abnormalities (such as skinhyperpigmentation), premature aging (such as hair greying, naildystrophy, oral leucoplakia), and anaemic pallor. Dokal, I. 2011.Hematology Am Soc Hematol Educ Program, 480-486. Thus an increase inoveral fitness can be determined by a decrease in physical attributesassociated with the particular condition exhibited by the treatedpatient. Overall fitness can also be measure by determining the bloodcount of the patient. In one embodiment, increased overall fitness ismeasured by determining the amount of leukocytes, lymphocytes,thrombocytes in a peripheral blood sample. Higher blood count indicatesan increased overall fitness. In certain embodiments, the blood count ina treated patient is increased by 5%, 10%, 15%, 20% or more, withreference to the blood count of an untreated patient suffering from thesame condition.

The efficacy of the treatment can also be measured by directlydetermining telomere length in sample taken from the patient. Telomerelength can be measured, for example, by using standard hybridizationtechniques, such as fluorescence in situ hybridization (FISH),Quantitative Fluorescent in situ hybridization (Q-FISH), or HighThroughput Quantitative Fluorescent in situ hybridization (HT Q-FISH).(Gonzalez-Suarez, Samper et al. 2001) in a sample taken from thepatient, Samples suitable for telomere analysis include bone marrowtissue and blood samples. Telomere length can also be measured asdescribed in Slagboom et al or Canela et al. (2007, PNAS 104:5300-5305).

In a particular embodiment, samples are taken from the patientundergoing treatment throughout the course of the treatment so that bothabsolute telomere length and the rate of telomere shortening over thecourse of treatment can be determined. Samples may be taken every dayduring the course of treatment, or at longer intervals. In oneembodiment, samples are taken once a week, once every two week, onceevery three weeks, once every 4 weeks, once every five weeks, once everysix weeks or longer.

Comparison of telomere length can be measured by a comparing theproportion of short telomeres in a sample taken from a patient. In oneembodiment, the proportion of short telomeres is the fraction oftelomeres presenting an intensity below the mean intensity of the sampleas measured by a in situ hybridization technique, such as FISH orQ-FISH. In embodiment, the proportion of short telomeres is the fractionof telomeres presenting an intensity 75%, 70%, 65%, 60%, 55%, 50%, 45%,40% or more below the mean intensity of the sample. In one particularembodiment, the proportion of short telomeres is the fraction oftelomeres presenting an intensity 50% or more below the mean intensityof the sample.

In another embodiments, the proportion of short telomeres is thefraction of telomeres below a certain length, e.g. 8 kb, 7 kb, 6 kb, 5kb, or shorter In one embodiment, the proportion of short telomeres isthe fraction of telomeres 8 kb or shorter. In another embodiment, theproportion of short telomeres is the fraction of telomeres 7 kb orshorter. In another embodiment, the proportion of short telomeres is thefraction of telomeres 6 kb or shorter. In another embodiment, theproportion of short telomeres is the fraction of telomeres 5 kb orshorter. In another embodiment, the proportion of short telomeres is thefraction of telomeres 4 kb or shorter. In another embodiment, theproportion of short telomeres is the fraction of telomeres 3 kb orshorter.

In one embodiment, the effectiveness of the treatment is measured by adecrease in the proportion of short telomeres in sample taken from atreated patient suffering from a condition associated with shorttelomere length as compared to a control sample. In one embodiment, theproportion of short telomeres in a sample taken from a treated patientis decreased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or greater ascompared to a control sample. In one embodiment, the control sample is asample taken from the same patient prior to the treatment, or taken atan earlier stage of the treatment. In another embodiment, the controlsample is a sample taken from a patient suffering from the samecondition and not provided the treatment.

In a further aspect, the invention is applied to the subject byadministering a pharmaceutical composition comprising an effectiveamount of any one of the gene therapy vectors compatible with theinvention described above.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

“Composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active. An “effective amount” is an amount sufficient toeffect beneficial or desired results. An effective amount can beadministered in one or more administrations, applications or dosages.

They will usually include components in addition to the active component(such as the gene therapy vector) e.g. they typically include one ormore pharmaceutical carrier(s) and/or excipient(s). A thoroughdiscussion of such components is available in Gennaro (2000) Remington:The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.

Compositions will generally be administered to a subject in aqueousform. Prior to administration, however, the composition may have been ina non-aqueous form. For instance, although some viral vectors aremanufactured in aqueous form, then filled and distributed andadministered also in aqueous form, other viral vectors are lyophilisedduring manufacture and are reconstituted into an aqueous form at thetime of use. Thus a composition of the invention may be dried, such as alyophilised formulation. The composition may include preservatives suchas thiomersal or 2-phenoxyethanol. It is preferred, however, that thecomposition should be substantially free from (i.e. less than 5 μg/mï)mercurial material e.g. thiomersal-free.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml e.g. about 10+2 mg/ml NaCl. Othersalts that may be present include potassium chloride, potassiumdihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride,calcium chloride, etc.

Compositions will generally have an osmolality of between 200 mOsm/kgand 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 290-310 mOsm/kg.

Compositions may include one or more buffers. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer (particularly with an aluminum hydroxide adjuvant); ora citrate buffer. Buffers will typically be included in the 5-20 mMrange.

The composition may include material for a single administration, or mayinclude material for multiple administrations (i.e. a ‘multidose’ kit).The inclusion of a preservative is preferred in multidose arrangements.As an alternative (or in addition) to including a preservative inmultidose compositions, the compositions may be contained in a containerhaving an aseptic adaptor for removal of material.

Compositions of the invention for use in humans are typicallyadministered in a dosage volume of about 0.5 ml, although a half dose(i.e. about 0.25 ml) may be administered to children.

As well as methods of treatment described herein, the invention alsoprovides a nucleic acid sequence encoding a TERT for use in therapy. Theinvention also provides a nucleic acid vector comprising a codingsequence for telomerase reverse transcriptase (TERT), for use in amethod of therapy and a gene therapy vector comprising a coding sequencefor telomerase reverse transcriptase (TERT), for use in a method oftherapy. In particular, the therapy may be treating or preventing acondition associated with short telomere length. As described formethods of treatment, the TERT nucleic acid sequence may be the sequenceas recited in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment or functionalequivalent thereof. The TERT protein may have a sequence as recited inSEQ ID NO: 2 or SEQ ID NO: 4, or a fragment or functional equivalentthereof.

The term “patient” refers to a mammal. In certain embodiments thepatient is a rodent, primate, ungulate, cat, dog, or other domestic petor domesticated mammal. In certain embodiments, the mammal is a mouse,rat, rabbit, pig, horse, sheep, cow, domestic cat or dog, or a human. Ina preferred embodiment, the patient is a human.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

EXAMPLES Example 1 Mouse Model for Dyskeratosis Congenita

Mice of C57B6 background that are homozygous carrier of a conditionalTRF1 transgene (TRF and further are transgenic for the Cre-recombinaseunder the control of the endogenous and interferon-inducible Mx1promoter will be used to test the efficacy of the telomerase genetherapy to treat Dyskeratosis congenital (DKC). To exclusively study theeffects of TRF1 ablation in the hematopoietic compartment bone marrowwill be transplanted from these mice into irradiated wild-type mice asdescribed previously (Beier et al., 2012). One month aftertransplantation the mice will be injected via their tail vein with4×10¹² AAV9 genomes carrying the mTERT cDNA under the control of thepotent cytomegalovirus promoter (for virus production, see below 3.3).By analogy, empty AAV9 lacking the telomerase gene will be injected intoa control group. Furthermore, to follow the viral tropism and transgeneexpression over time, a separate group of animals will be injected withAAV9-eGFP. One week after the virus infection TRF1 deletion in the bonemarrow will be induced by long-term polyinosinic-polycytidylic acid(pI:pC) treatment with intraperitoneal injections every third day. pI:pCacts as immunostimulant and activates Cre expression which in turn leadsto TRF1 deletion in approximately 50% of hematopoietic cells (upon eachinjection) with the above mentioned consequences (see 2). In contrast topI:pC treatment, animal groups previously infected with AAV9-mTERT andAAV9-empty will not undergo pI:pC treatment to serve as additionalcontrol cohorts.

With this experimental design the dramatic telomere shortening, owed tocompensatory proliferation in the remaining hematopoietic cells whichhave not lost TRF1, by ectopic expression of telomerase, will bereduced. The strongest measure to assess the effectiveness of the genetherapy is an extended life span by virtue of delayed or prevented bonemarrow failure (end point of experiment=death of animals). Furthermore,successful telomerase treatment should show improvements with regards tooverall fitness of the animals, i.e. no skin abnormalities and noanaemic pallor. The latter goes hand in hand with higher blood counts(leukocytes, lymphocytes, thrombocytes), which will be determined fromperipheral blood samples. The efficacy of telomerase expression on themolecular level will include telomere length measurements. To so Q-FISHanalysis from bone marrow tissue sections and high throughput Q-FISHanalysis from peripheral blood samples will be performed. For thesecond, blood will be taken every three to four weeks throughout thecourse of the experiment. In this way not only absolute telomere length,but also the rate of telomere shortening over time can be determined.Moreover, attenuated or abolished replicative senescence and exhaustionof stem and progenitor cells in the hematopoietic compartment will bemonitored by assessment of common senescent markers such asbeta-galactosidase activity and p21 protein levels. These markers aswell as γH2AX and phospho-CHK1, molecular markers for replicativestress, can then be correlated with telomere length and survival of theanimals.

Example 2 Production of Viruses

AAV based viral vectors for transduction will be generated by tripletransfection of HEK293T cells as described in (Matsushita et al., 1998).Briefly, to 80% confluence grown cells are co-transfected with plasmids(1) carrying the expression cassette flanked by the AAV9 viral ITRs, (2)a helper plasmid carrying the AAV rep2 and cap9 genes, and (3) a plasmidcarrying the adenovirus helper functions. The expression cassettesharbour murine TERT under the control of CMV promoter plus 3′-UTR(AAV9-mTERT), CMV promoter (AAV9-empty) alone and eGFP under the controlof CMV promoter and SV40 polyA signal (AAV9-eGFP). Vectors are purifiedfollowing an optimised method based on two consecutive cesium chloridegradients (Ayuso et al., 2010). Titres of viral genomes particles aredetermined by quantitative real time PCR. Viruses can be stably kept at−80° C. until infection of animals.

Example 3 Telomere Analysis

Telomere Q-FISH Analysis on Paraffin Sections

Q-FISH determination on paraffin-embedded tissue sections mice arehybridized with a PNA-telomeric probe, and fluorescence intensity oftelomeres are determined as described (Gonzalez-Suarez, Samper et al.2001). Quantitative image analysis is performed using the DefiniensDeveloper Cell software (version XD 1.2; Definiens AG). For statisticalanalysis a two-tailed Student t test is used to assess significance(GraphPad Prism software).

Quantitative Real-Time RT-PCR

Total RNA from tissues is extracted with Trizol (Life Technologies). RNAsamples are DNase I treated and are used as template for a reversetranscription reaction using random primers and Superscript ReverseTranscriptase (Life

Technologies), according to the manufacturer's guidelines. Quantitativereal-time PCR is performed using an ABI

PRISM 7700 (Applied Biosystems), using DNA Master SYBR Green I mix(Applied Biosystems).

The primers: Actin-For: (SEQ ID NO: 7) GGCACCACACCTTCTACAATG; Actin-Rev:(SEQ ID NO: 8) GTGGTGGTGAAGCTGTAG; TERT-For: (SEQ ID NO: 9)GGATTGCCACTGGCTCCG; TERT-Rev: (SEQ ID NO: 10) TGCCTGACCTCCTCTTGTGAC.p16-For: (SEQ ID NO: 11) CGTACCCCGATTCAGGTGAT p16-Rev: (SEQ ID NO: 12)TTGAGCAGAAGAGCTGCTACGT Axin2-For: (SEQ ID NO: 13) GGCAAAGTGGAGAGGATCGACAxin2-Rev: (SEQ ID NO: 14) TCGTGGCTGTTGCGTAGG Cyclin D1 - For:(SEQ ID NO: 15) TGCGCCCTCCGTATCTTAC Cyclin D1 - Rev: (SEQ ID NO: 16)ATCTTAGAGGCCACGAACATGC CD44 - For: (SEQ ID NO: 17)CAGCCTACTGGAGATCAGGATGA CD44 - Rev: (SEQ ID NO: 18)GGAGTCCTTGGATGAGTCTCGA KIf4 - For: (SEQ ID NO: 19)GCGAACTCACACAGGCGAGAAACC KIf4 - Rev: (SEQ ID NO: 20)TCGCTTCCTCTTCCTCCGACACA Tieg1 - For: (SEQ ID NO: 21) CCCATTGCCCCTGCTCCTGTieg1 - Rev: (SEQ ID NO: 22) TGTGTCCGCCGGTGTCTGG

Statistical analyses (Student's t-test) is performed on the Ct values asdescribed before (Munoz, Blanco et al. 2005).

Example 4 Telomerase Gene Therapy in Aplastic Anemia

Mice and Animal Procedures

Mice were of pure C57/BL6 background and were produced and housed at thespecific pathogen-free (SPF) animal house of the CNIO in Madrid, Spain.Trfl^(lox/lox) Mx1-Cre and Trfl^(lox/lox) Mx1-wt mice were generatedpreviously described (Martinez et al., 2009) ENREF 20. For bone marrowtransplantation 10 weeks old Trfl^(lox/lox) Mx1-Cre mice were used asbone marrow donors for transplantation into 8 weeks old lethally (12Gy)irradiated wild-type mice as previously described (Beier et al., 2012,Samper et al., 2002). A total of 2 million cells were transplanted viatail vein injection at a donor:recipient ratio of 1:8 and mice were leftfor a latency period of 30 days to allow bone marrow reconstitution. Toinduce Cre expression, mice were intraperitoneally injected withpolyinosinic-polycytidylic acid (pI:pC; Sigma-Aldrich) (15 ug/g bodyweight) 3 times per week for a total duration of 5 weeks. Mice were leftfor an additional week before they were randomly assigned to two groupsfor the treatment with AAV9-Tert or AAV9-empty gene therapy vectors.Vectors were administered via tail vein injection at a concentration of4×10E12 viral genomes per mouse.

Gene Therapy Vector Production

Viral vectors were generated as described previously (Matsushita et al.,1998) and purified described in (Ayuso et al., 2010). Briefly, vectorswere produced through triple transfection of HEK293T. Cells were grownin roller bottles (Corning, N.Y., USA) in Dulbecco's Modified Eagle'sMedium supplemented with FBS (10% v/v) to 80% confluence and thenco-transfected with: plasmid-1 carrying the expression cassette for geneof interest flanked by the AAV2 viral ITRs; plasmid-2 carrying the AAVrep2 and cap9 genes; plasmid-3 carrying the adenovirus helper functions(plasmids were kindly provided by K. A. High, Children's Hospital ofPhiladelphia). Expression cassettes were under the control of thecytomegalovirus (CMV) promoter and contained a SV40 polyA signal forEGFP and the CMV promoter and the 3′UTR of the Tert gene as polyA signalfor Tert. AAV9 particles were purified following an optimized methodusing two caesium chloride gradients, dialysed against PBS, filtered andstored at −80° C. until use (Ayuso et al., 2010). Viral genomesparticles titres were determined by a standardized quantitative realtime PCR method (Ayuso et al., 2014) and primers specific for the CMVsequence:

CMV-Forward: (SEQ ID NO: 23) 5′-CAATTACGGGGTCATTAGTTCATAGC; CMV-Reverse:(SEQ ID NO: 24) 5′-ATACGTAGATGTACTGCCAAGTAGGA.

Histology

Bone marrow samples (sternum or tibia bone) were fixed inphosphate-buffered 4% formaldehyde and bones after decalcificationparaffin embedded. 5 μm tissue sections were stained withHematoxylin-Eosin for histological bone marrow assessmentImmunohistochemistry was performed on deparaffinized tissues sections.After antigen retrieval samples were processed with the anti-EGFPantibodies (rabbit anti-EGFP, 1:200; Abcam, ab290). EGFP positive cellswere counted in a semi automated way using ImageJ software.

FACS Sorting

For sorting of HSCs whole bone marrow cells were extracted from the longbones (femur & tibia) as previously described (Samper et al., 2002).Erythrocytes were lysed by incubating cells for 10 min in 10 mlerythrocyte lysis buffer (Roche), washed once with 10 ml PBS, andresuspended in FACS buffer (PBS, 2 mM EDTA, 0.3% BSA) containingFc-block (1:400) at a concentration of 5-10×10{circumflex over ( )}6cell/100 μl. Cells were incubated for 10 min and washed once in FACSbuffer. Cells were then resuspended in FACS buffer at20-25×10{circumflex over ( )}6 cell/ml and the antibody cocktail wasadded as follows: Anti-sca-1-PerCP-Cy5.5 (1:200), lin cocktail-eFluor450(1:50) (all eBioscience), and anti-c-kit-APC-H7 (1:100) (BD Pharmingen).Cells were incubated for 30 min. After washing cells twice with PBS, 2 Lof DAPI (200 g/mL) was added and cells were subsequently sorted in aFACS ARIA IIu (Becton Dickinson, San Jose, Calif.) into HSCs (linnegatice, scal and c-kit positive) and lineage positive (lin positive)fractions.

Colony Forming Assay

Short-term colony-forming assay (CFA) was performed by plating 1×10⁴ and2×10⁴ freshly isolated mononucleated bone marrow cells (erythrocyteswere lysed as described above) in 35-mm dishes (StemCell Technologies)containing Methocult (methylcellulose-based) media (StemCellTechnologies) as described in the manufacturer's protocol. Allexperiments were performed in duplicates and the numbers of coloniesformed were counted after 12 days incubation at 37° C.

Blood Counts

Peripheral blood was drawn from the facial vein (˜50 μl) and collectedinto anti-coagulation tubes (EDTA). Blood counts were determined usingan Abacus Junior Vet veterinary hematology analyzer.

Quantitative Real-Time PCR and Western Blots

Total RNA from whole bone marrow extracts or FACS sorted bone marrowcells was isolated using Qiagen's RNeasy mini kit according to themanufacturer protocol. The optional DNaseI digest was always performed.Quantitative real-time PCR was performed using an ABI PRISM 7700 orQuantStudio 6 Flex (both Applied Biosystems). Primers sequences for Tertand reference genes Act1 and TBP are as follows:

Tert-Forward (SEQ ID NO: 9) 5′GGATTGCCACTGGCTCCG; Tert-Reverse(SEQ ID NO: 10) 5′TGCCTGACCTCCTCTTGTGAC; Actin-Forward (SEQ ID NO: 7)5′GGCACCACACCTTCTACAATG; Actin-Reverse (SEQ ID NO: 8)5′GTGGTGGTGAAGCTGTAG; TBP-Forward (SEQ ID NO: 25)5′CTTCCTGCCACAATGTCACAG; TBP-Reverse (SEQ ID NO: 26)5′CCTTTCTCATGCTTGCTTCTCTG.

Q-FISH Telomere Analysis

Q-FISH analysis on bone marrow tissues sections was performed asdescribed previously (Samper et al., 2000). Briefly, tissues sectionswere post fixed in 4% formaldehyde for 5 min, washed 3×5 min in PBS andincubated at 37° C. for 15 min in pepsin solution (0.1% Porcine Pepsin,Sigma; 0.01M HCl, Merck). Washes and fixation was repeated and slidesdehydrated in a 70%-90%-100% ethanol series (5 min each). Slides were 10min air-dried and 30 μl of telomere probe mix added to each slide (10 mMTrisCl pH 7, 25 mM MgCl2, 9 mM citric acid, 82 mM Na2HPO4, 70% deionizedformamide (Sigma), 0.25% blocking reagent (Roche) and 0.5 mg/mlTelomeric PNA probe (Panagene)), a cover slip added and slides incubatedfor 3 min at 85° C., and 2 h at room temperature in a wet chamber in thedark. Slides were washed 2×15 min in 10 mM TrisCl pH 7, 0.1% BSA in 70%formamide under vigorous shaking, then 3×5 min in TBS 0.08% Tween20, andthen incubated in a 40,6-diamidino-2-phenylindole (DAPI) bath (4 mg/ml 1DAPI (Sigma) in PBS). Samples were mounted in Vectashield (Vector™).Confocal image were acquired as stacks every 0.5 μm for a total of 1.5μm using a Leica SP5-MP confocal microscope and maximum projections weredone with the LAS-AF software. Telomere signal intensity was quantifiedusing Definiens software.

High throughput (HT)-Q-FISH on peripheral blood leukocytes was done asdescribed (Canela et al., 2007a). Briefly, 120-150 μl of blood wereextracted from the facial vein. Erythrocytes were lysed (Erythrocytelysis buffer, Qiagen) and 30-90 k leukocytes were plated in duplicateinto clear-bottom, black-walled 96-well plates pre-coated for 30 minwith 0.001% poly-L-lysine. Plates were incubated at 37° C. for 2 h andfixed with methanol/acetic acid (3:1, v/v) 2×10 min and overnight at−20° C. Fixative was removed, plates dried for at least 1 h at 37° C.and samples were rehydrated in PBS. Plates were then subjected to astandard Q-FISH protocol (see above) using a telomere-specific PNA-CY3probe; DAPI was used to stain nuclei. Sixty images per well werecaptured using the OPERA (Perkin Elmer) High-Content Screening system.TL values were analysed using individual telomere spots (>10,000telomere spots per sample). The average fluorescence intensities of eachsample were converted into kilobase using L5178-R and L5178-S cells ascalibration standards, which have stable TLs of 79.7 and 10.2 kb,respectively. Samples were analyzed in duplicate.

AAV9-Tert Targets Bone Marrow and Hematopoietic Stem Cells

First, we set out to address the capability of AAV9 vectors to transducethe bone marrow upon intra-venous injection by using both a AAV9-EGFPreporter virus, which allows determination of the location andpercentage of AAV9-transduced cells, as well as by determining Tert mRNAexpression in vivo in different bone marrow cell populations followingAAV9-Tert treatment. To this end, we first injected wild-type mice withAAV9-EGFP particles at a concentration of 3.5E12 viral genomes per mousethrough tail vein injections Immunohistochemistry analysis of bonemarrow section with specific anti-EGFP antibodies revealed 2% positiveEGFP expressing cells in the middle bone sections and this was increasedup to 10% in the regions adjacent to the joints, which were the onesshowing the highest AAV9-transduction. We then injected wild-type micewith the same amount of AAV9-Tert particles and determined Tert mRNAexpression by RT-PCR in whole bone marrow isolates at two weeks and 8months after virus injection. As soon as two weeks post-treatment withthe AAV9 vectors, we found increased Tert mRNA expression in theAAV9-Tert treated mice compared to those treated with the AAV9 emptyvector and this difference was maintained still 8 months after initialtreatment. We then studied Tert mRNA expression specifically in theblood-forming cells of the bone marrow. To this end, we performed FACSsorting of c-kit and Sca-1 positive HSCs cells and lin-positive lineagecommitted cells. We found a significant increase in both HSCs (10 fold)and lineage committed bone marrow cells (3.5 fold) in Tert mRNA inAAV9-Tert treated mice compared to mice treated with the empty vector,demonstrating that bone marrow cells including HSCs cells are targetedby Tert gene therapy. Given that we achieved increased Tert expressionin HSCs, we next addressed whether this affected their stem cellpotential. To this end, we performed a colony forming cell assay(MethoCult) Interestingly, we observed significantly increased number ofcolonies in the AAV9-Tert mice compared to the empty vector controls.

In summary these data suggest that AAV9 administered at a high dose cantarget hematopoietic cells and that these enhances the proliferationcapacity of those cells.

AAV9-Tert Treatment in a Mouse Model of Aplastic Anemia Rescues Survival

We next tested whether treatment with AAV9-Tert is effective inincreasing survival upon induction of lethal aplastic anemia owing tocritically short telomeres (Beier et al., 2012). In particular, we useda conditional Trfl mouse model recently developed by us in which welethally irradiate wild-type mice and transplant them with bone marrowisolated from Trfl^(lox/lox) Mx1-Cre mice to exclusively study theeffects on bone marrow. Trfl deletion can be induced by administrationof pl:pC and subsequent expression of the Cre recombinase (Beier et al.,2012). Cells depleted for Trfl die and are rapidly removed from the bonemarrow, while cells that remain with intact Trfl undergo compensatoryrounds of cell division which leads to rapid telomere shortening, followby replicative senescence and finally results in bone marrow failure. Inthe specific experimental settings here we induced Trfl deletion byinjecting mice 3 times per week with pI:pC for a total period of 5weeks, at which point these mice start showing signs of aplastic anemia(Beier et al., 2012). One week after we stopped the induction of Trfldeletion, mice were subjected to gene therapy treatment with AAV9-Tertor AAV9-empty control vectors. We monitored the survival of these micefor 100 days following the treatment with the AAV9 vectors. Strikingly,AAV9-Tert treatment drastically improved survival (87%) compared withmice treated with the empty vector (55%) (FIG. 1A). In particular, whileonly 4 mice injected with AAV9-Tert developed aplastic anemia duringthis time (13%), 16 mice of the control group (44%) died with clearsigns of aplastic anemia (FIG. 1B,C). In agreement with the anemicappearance blood count analysis from these mice (blood drawn fromAAV9-empty and AAV9-Tert upon sacrificing) showed a drastic drop inplatelet count and haemoglobin level compared with mice without signs ofaplastic anemia (FIG. 1D, E). Post mortem histopathologic analysis ofbone marrow sections from mice that died during the first 100 daysfurther confirmed the aplastic anemia phenotype. In particular, micepresented with severe bone marrow hypo- and aplasia in 2 or all 3 bloodlineages. While the diagnosis at the point of death in both groups wasmarrow bone failure and aplasia the phenotype appeared milder in theAAV9-Tert group compared with the AAV9-empty group as seen by higherbone marrow cellularity.

Our results suggest that AAV9-Tert gene therapy significantly reducesaplastic anemia mortality by preventing the loss of blood forminghematopoietic cells.

Telomerase Treatment Leads to Telomere Elongation in Peripheral Bloodand Bone Marrow

Because the aplastic anemia phenotype in our mouse model is caused bythe loss of telomeres, we next compared telomere length in mice treatedwith telomerase to mice receiving the control vector. First we usedHT-Q-FISH technology (Canela et al., 2007b) to follow telomere length inperipheral blood monocytes in a longitudinal manner. To do so, weextracted blood at 4 different time points; after bone marrowengraftment (1), after pI:pC treatment (2), 2 months after AAV9injection (3) and 4 months after AAV9 injection (4). As expected wefound that telomere length between time point 1 and 2 in both groupsdrops by approximately 10 kb which is owed to the pI:pC treatment. Whiletelomere length in the AAV9-empty group between time point 2 and 4continuous to slightly shorten, AAV9-Tert treatment led to a netincrease in average telomere of 10 kb (FIG. 2A,B). Throughout the courseof this experiment AAV9-empty treated mice showed an average telomerelength loss of 12 kb, whereas in AAV9-Tert treated mice telomeres werere-elongated to similar levels as before the pI:pC treatment (FIG. 2C).Next we performed Q-FISH analysis on bone marrow cross-sections. Inagreement with longer telomere length in peripheral blood we found thatAAV9-Tert treated mice had significantly longer telomeres compared withempty vector treated mice (FIG. 2D, E).

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1.-12. (canceled)
 13. A method of treating a condition associated with adisease, the method comprising: administering a recombinant viral vectorcomprising at least a capsid and a nucleic acid comprising a codingsequence for telomerase reverse transcriptase (TERT) to a subject,wherein the condition is associated with pulmonary fibrosis.
 14. Themethod of claim 13, wherein TERT is encoded by a nucleic acid sequencecomprising a sequence that is at least 60% identical to the sequence ofSEQ ID NO: 1 or SEQ ID NO:
 3. 15. The method of claim 13, wherein TERTcomprises an amino acid sequence that is at least 60% identical to theamino acid sequence of SEQ ID NO:2 or SEQ ID NO:
 4. 16. The method ofclaim 13, wherein the nucleic acid sequence encoding TERT is operablylinked to a regulatory sequence that drives the expression of the codingsequence.
 17. The method of claim 13, wherein the vector is anon-integrative vector.
 18. The method of claim 13, wherein the vectoris an adeno-associated virus-based non-integrative vector.
 19. Themethod of claim 13, wherein the vector is an adeno-associatedvirus-based vector whose capsid is derived from a serotype 9adeno-associated virus (AAV9).
 20. The method of claim 19, wherein thenucleic acid sequence packaged in the capsid is flanked at both ends byinternal terminal repeats of the serotype 2 adeno-associated virus. 21.The method of claim 13, wherein the vector comprises a regulatorysequence which is a constitutive promoter.
 22. The method of claim 21,wherein the constitutive promoter is the cytomegalovirus (CMV) promoter.23. The method of claim 13, wherein the pulmonary fibrosis ischaracterized by mutations in a gene or genes involved in telomeremaintenance.